The Antitoxins 129 



2. Determine accurately the least quantity of the serum that will protect 

 the guinea-pig against ten times the above determined least fatal dose of toxin. 



3. Express the required dose of antitoxic serum as a fraction of a cubic centi- 

 meter and multiply by 10; the result is one unit. 



Example: It is found that o.oi cc. of a toxic bouillon kills at least 9 out of 10 

 guinea-pigs, and is therefore the least certainly fatal dose. Guinea-pigs receive 

 ten times this dose of the toxic bouillon plus varying quantities of the serum to 

 be tested, measured by dilution say Hooo cc., Hsoo cc., 3^000 cc. The 



lied b 



first two live. The fraction >1}500 is now multiplied by 10; ^3500 X 10 



= i unit. So we find that each cubic centimeter of the serum contains 250 



units. 



This method would be satisfactory were it not for certain variations in the 

 toxic bouillon by which the strength is worked out. Ehrlich,* in an elaborate 

 investigation of these changes, has clearly proved that an ever-changing toxin 

 cannot be a satisfactory standard, because it does not possess uniform combining 

 affinity for the antitoxin. He shows by a labored scheme that the toxicity of 

 the bouillon is no index to its antitoxin-combining power, which, of course, must 

 be the foundation of the test. The toxin, under natural conditions, is changed 

 with varying rapidity into toxoids, of which he demonstrates three groups 

 prototoxoids, syntoxoids, and epitoxoids. The epitoxoids have a greater anti- 

 toxin-combining power than the toxin itself, yet have no toxic action upon the 

 guinea-pigs, hence cause confusion in the results. 



To secure a satisfactory measure of the antitoxic strength of a serum, it is 

 therefore more important to first determine the antitoxin-combining power of 

 the toxin or toxic bouillon to be used than to determine its guinea-pig fatality, 

 and this is what Ehrlich endeavors to do. 



(B) Ehrlich' s Method. In this method the unit is the same as in Behring's 

 method, but its determination is arrived at by a very important modification of 

 the method, by which the standard of measurement is a special antitoxin of 

 known strength, by which the antitoxin-combining power of the test toxic bouil- 

 lon is first determined. Ehrlich began by determining the antitoxic value of a 

 serum as accurately as possible by the old method, and then used that serum as 

 the standard for all further determinations. The serum was dried in a vacuum, 

 and two grams of the dry powder were placed in each of a large number of 

 small vacuum tubes, connecting with a small bulb of phosphoric anhydride. 

 In this way the standard powder was protected from oxygen, water, and other 

 injurious agents by which variations in its strength could be initiated. Periodic- 

 ally one of these tubes was opened and the contained powder dissolved in 200 

 cc. of a mixture of 10 per cent, aqueous solution of sodium chloride and glycerin. 

 The subsequent calculations are all based upon the strength of the antitoxin 

 powder. In Ehrlich's first test serum i gram of the dry powder represented 

 1700 units. Of the solution mentioned, i cc. represented 17 units; ^ 7 cc., 

 one unit. 



Having by dilution i cc. of the first dilution in 17 of water secured the 

 standard unit of antitoxin in a convenient bulk for the subsequent manipulations, 

 it is mixed with varying quantities of the toxic bouillon to be used for testing the 

 new serums, until the least quantity is determined that will cause the death of a 

 250 gram guinea-pig in exactly four days, when carefully injected beneath the 

 skin of the animal's abdomen. This quantity of toxin is the test dose. If the 

 toxic bouillon was "normal" in constitution, it should represent 100 of the least 

 certainly fatal doses that formed the basis of the old method of testing, but as 

 toxic bouillons contain varying quantities of toxoids it may equal anywhere 

 from fifty to one hundred and fifty times that dose. 



The test dose of toxic bouillon, having been determined, remains invariable 

 throughout the test as before, the serum to be tested for comparison with the 

 standard being modified. The calculation is, however, different because the 

 guinea-pig is receiving, not ten times, but more nearly one hundred times the 

 least fatal dose, and the quantity of the antitoxic serum that preserves life 

 beyond the fourth day is itself the unit. 



Example: The sample of serum issued as the standard contains 17 units per 

 cubic centimeter. Serum i cc. + water 16 cc. = i cc. is the unit, i cc. of 

 the dilution containing one antitoxic unit is mixed with o.oi, 0.025, 0.05, 0.075, 

 o.i cc. of the toxic bouillon. All the animals receiving less than o.i cc. live. 



* "Klinisches Jahrbuch," 1897. 



